Floxed Allele

Facts about Producing a Floxed Allele to Create Complete Knockouts

An allele is an alternative form of a specific gene. While some genes are only present once in the body, depending on the species and the particular gene in question, others have two or more alleles of the same gene. This can result in various phenotypic traits, such as different colored eyes, skin or hair.

In humans, as in the case of most multicellular organisms, each gene has two alleles of the same gene, which are associated with the two sets of chromosomes they have. In many cases, subtle mutations occur in germ cells, which then pass on the mutated allele to the following generation, leading to physiological changes that drive the species’ evolution. These mutations can also cause harmful genetic conditions.

Genetic researchers study the production of floxed alleles in animal models, such as mice, to learn more about these genetic mutations. Because the mouse genome is significantly close to that of humans, it’s an excellent model to study these conditions. Using the Cre/lox recombination system, together with the generation of a floxed allele, scientists can create complete knockout mice.

The production of knockout mouse models that target a specific mutated allele is essential for new experiments to study genetic disorders. Because some diseases impair a certain gene by generating mutated alleles that nullify it, creating knockout mouse models of that particular gene might be necessary to observe the effects that eliminating the gene may have in vivo. At the same time, certain mutations can be more likely to cause genetic disorders than others. By using floxed alleles and knockout mice, researchers can better identify these mutations.

RELATED: Conditional Knockout Mouse Models

Floxed Alleles in Animal Models

• Used to make deletion of genes in site and time-specific manner
• Flank gene of interest with LoxP sites
• Inject a plasmid or cross into strain that expresses Cre recombinase where/when/how you want loss-of-function to occur

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Lesser Known Facts about Cre/lox Recombination and Floxed Allele

Anyone who plans to use the Cre/lox recombination system to produce a floxed allele and knockout mice should be aware of a few important and lesser-known facts that experts tend to point out.

1. The Cre/lox system is more efficient in converting floxed alleles to complete knockouts if the alleles are passed down from the mother. While this isn’t always the case, and the parental inheritance pattern doesn’t matter for all Cre strains, it is always best to compare maternal and paternal transmission results when possible.

2. It’s actually possible to generate a knockout from a floxed allele even if the mouse doesn’t necessarily carry a Cre transgene. This option is prevalent in the case of Vasa-Cre strains, where the Cre mRNA protein is expressed even when the transgene is not present within the oocyte. Strains that feature maternal Cre expression in the oocyte can help save a significant amount of time during the process of converting floxed alleles to knockouts, since no additional breeding will be needed in order to eliminate the Cre strain after the recombination process is confirmed.

3. Cre recombinase can be toxic in ES cells, causing difficulties when linking expression to genes that are active during embryonic time points. Random integration transgenic mice produced via pronuclear injection have been used as an alternative to avoid embryonic stem cell lethality; however, these models do not faithfully reflect endogenous gene expression patterns. To solve this problem, an approach is needed that enables native gene expression patterns while avoiding ES cell toxicity.

4. To eliminate concerns about ES cell toxicity, you can generate your mouse line by splitting Cre recombinase in half using a FRT-flanked neomycin selection cassette. This split design can be used for gene replacement or co-expression knockin, generating Cre recombinase expressing mouse lines that follow the native expression patterns of any target gene, regardless of expression location or timepoints.

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